15 - immunoassays Flashcards
2 methods of using antibodies in diagnostics
tracking pathogens in their natural environment
looking at what circulating biomarkers are present in the blood
qualitative measurements
allow you to look at where things are distributed
quantitative measurements
allow you to measure how much antigen is present
qualitative immunoassay formats (4)
qualitative: Ouchterlony Immunodiffusion Immunofluorescence Immunoelectron gold microscopy Western Blotting
ouchterlony immunodiffusion (OID)
detects antibody-antigen interactions
allows indentification of similarities between antigenic properties of two unknown solutions
indication of +ve OID test
precipitin lines seen in agar gel
method of OID
place both into a plate of agar and allow them to diffuse towards each other
uses of OID test
forms basis of allergen tests
Has the patient got circulating antibodies against an allergen?
Is there more than one source of allergen causing the problem?
why is formation of immune complexes (antigen-antibody) important
larger bodies are easier for macrophages to destroy
problem with too little antigen in OID testing
antibody concentration too high
immune complexes cannot form
no agglutination
no precipitation
problem with too much antigen in OID testing
antibody concentration too low
antigen excess
steric hinderance
immune complexes cannot form
in OID, if precipitin lines fuse completely into 1 smooth arc…
then the test has shown the presence of
identical antigenic determinants in the sample
in OID, if the precipitin lines cross but without any interaction…
then the two solutions dont share any common determinants detectable by antiserum
they react but are not related
OID:
if the precipitin lines fuse, but an additional spur forms towards the well containing solution…
the the two solutions share some common determinants but solution A has additional unique determinant detected by the antiserum
western blotting
means of looking at antigenic similarity
specific antibodies used to identify particular antigens in a mixture of proteins that have been resolved and transferred to a membrane
protein separation technique in western blotting
SDS-PAGE
method of western blotting
separation of proteins from mixture using SDS-PAGE
transfer by electroblotting
incubate with specific antibodies for the target antigen
unbound antibodies washed away
location of bound antibodies identified using colour marker
what does PAGE stand for
POLY-ACRYLAMIDE GEL ELECTROPHORESIS
speed of migration of in an electrical field depends on…
the dimension, form and charge of the molecules
high molecular weight proteins found at the top of the gel
use of SDS
provides molecules with negative charge
so charge does not affect separation
why do we denature proteins in western blotting
to break disulphide bridges
SDS-PAGE in 1D
one dimension of separation
all given same -ve charge, proteins separate according to molecular weight
SDS-PAGE in 2D
2 dimensions of separation:
1- separate proteins on basis of pH - according to charge
2 - separation according to molecular weight - SDS electrophoresis
use of secondary antibody in western blotting
used as a recording molecule
e. g.
- conjugate anti-mouse antibody to fc domain of primary antibody that is being inserted and tested
- enzyme on secondary antibody will convert substrate to a different colour
Scedosporium prolificans
An ‘emerging’ fungal pathogen of immuno-compromised/AIDs patients
source is often pot plants
problems with Scedosporium prolificans
resistant to many anti-fungal drugs
causes lesions in CNS of those infected
virulence factor of Scedosporium prolificans
heavy melanisation
gene disruption removes enzyme to make black pigment –> reduced virulence
immunofluorescence
qualitative and widely used technique
can be direct or indirect
antibodies get tagged with fluorescent reagents
apply UV: reagents fluoresce at different absorbances
examples of fluorescent reagents
fluorochromes
FITC: Fluorescein IsoThioCyanate (green)
TR: Texas Red
immunofluorescence uses
detect localisation of molecules of interest –> e..g distribution of an antigen
identify pathogenic organisms in tissues
importance of clusters of differentiation (CD) in immunofluorescence
antibodies tell you where the relevant CD is localised on outside of immune cell
indirect immunofluorescnce
use of secondary antibody which is conjugated to fluorochrome
secondary antibody binds to Fc domain of primary antibody which bins to antigen
direct immunofluorescnce
fluorochrome attached directly onto antibody of interest
immuno-electron gold microscopy
qualitative technique
antibodies tagged with gold particles (instead of fluorochrome) are used to detect molecules of interest
uses/importance of immuno-electron gold microscopy
- allows localisation of antigen-antibody interactions at the nm scale (better resolution)
- powerful method of visualising intracellular antigens
and organelles
importance of fungal antigen
only produced when pathogen is in active germination or polarisaed (hyphal) growth
good biomarker
ELISA:
Enzyme-linked Immunosorbent Assays
quantitative technique
detects antigens/antibodies using enzyme-substrate reactions
alows high throughput screening of antibodies
how are the enzymes coupled to the antibodies in ELISA
either directly (to the antibody) or indirectly (to the anti-immunoglobulins)
2 main ELISA formats
- Plate-Trapped-Antigen (PTA)-ELISA
2. Double-Antibody-Sandwich (DAS)-ELISA
example of anti-immunoglobulin use in ELISA
. Goat anti-mouse peroxidase conjugate
how is quantification in ELISA achieved
using standard calibration curves
known amount of antigen on plate and colour reaction
what separates IgG from IgM
different heavy chains
IgM is a pentamer and is therefore really good at pulling down the antigen
fusarium
common plant pathogen
can infect humans
e.g. fusarium nail infection
PTA-ELISA
plate trapped antibody -ELISA
results of PTA-ELISA of fusarium
probe with specific antibody
absorbance increases over time where fungus is present and antibdy binds
Rhizoctonia solani
Ubiquitous soil-borne plant pathogen
worldwide distribution and wide host range
prevents any growth in soil if infected
Difficult to control using chemicals
why is Rhizoctonia solani difficult to control using chemicals
- Soil dwelling
- Widespread resistance to fungicides
- Soil fumigants are banned (Class II ozone depletors)
what is the most effective way to deal with Rhizoctonia solani
to ‘evade’ the pathogen altogether
do not use in contaminated soils
test soils for infestation prior to sowing with susceptible crops
R. solani-specific mAbs
monoclonal antibdies raised specifically against the fungal pathogen - Rhizoctonia solani
antigen is secreted by live cells only
examples of R. solani-specific mAbs
mAb EH2 - protein epitope
mAb EE1 - carbohydrate epitope
procedure to diagnose Rhizoctonia solani
Stimulate active growth
using baits
- Detect R. solani using specific
mAbs - Recover positive isolates for
further testing
method to construct a baiting molecule
Take soil sample and put in petri dish with lid
Bait used to bait pathogen out of the soil
Colonise bait to detect pathogen using monoclonal antibodies
Coat microtitre plate with 1st antibody
Bathing solution
Pathogen present will secrete antigen
Process double antibody ELISA
mass extinctions of what seen
amphibians
Examples = frogs, toads, salamanders
hypervirulent lineage of fungus
causing extinction of amphibian species
500 species of frog lost over last 10 years
chytrid fungus (Bd)
aquatic, motile fungus that causes severe disease in amphibians.
causes chronic disease of the skin –> Chytridiomycosis
life cycle of chytrid fungus (Bd)
lasts 4-5 days
starts as tiny zoospore that swims around until burrowing and penetrating skin of host
develops into a thallus which matures into a sporangium
grows and bursts to release more zoospores, chronic re-infection or infection of other organisms
Chytridiomycosis
Colonises keratinised skin layer.
Hyperplasia + Hyperkeratosis = Disrupted electrolyte balance
Ultimately leads to cardiac arrest and death.
amphibians require skin to breathe and drink
hyperplasia
enlargement of organ tissue due to cellular proliferation.
hyperkeratosis
thickening of the skin
diagnosis of chytridiomycosis
difficult due to ambiguous symptoms
qPCR is gold standard
histology important
lateral flow assay
similar to a pregnancy test dipstick assay
2 lines appear if you infected with disease of interest
semi-quantitative point-of-care test
quantitative immunoassay formats (2)
quantitative:
PTA-ELISA and DAS-ELISA
Lateral-Flow Assay
